S H 

67 




Book 'A 



DEPARTMENT OF COMMERCE 

BUREAU OF FISHERIES 

HUGH M. SMITH, Commissioner 



MORTALITY IN PIKE-PERCH EGGS 



By 

FRANZ SCHRADER 

Formerly Fish Pathologist, U. S. Bureau of Fisheries 

and 

SALLY HUGHES SCHRADER 

Temporary Assistant, U. S. Bureau of Fisheries 



APPENDIX V TO THE REPORT OF THE U. S. COMMISSIONER 

of Fisheries for 1922 




Bureau of Fisheries Document No. 926 



PRICE, 5 CENTS 

Sold only by the Superintendent of Documents, Government Printing Office 

Washington, ID. C. 



WASHINGTON 

GOVERNMENT PRINTING OFFICE 

1922 



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RECEIVED 

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DOCUMENTS UiV.aslQN 






MORTALITY IN PIKE-PERCH EGGS IN HATCHERIES. 1 



By Franz Schkadek, formerly Fish Pathologist, U. 8. Bureau of Fisheries, 

and 
Sai.i.y Htjghes Schrader, Tern pora ry Assistant, U. S. Bureau of Fisheries. 



The remarkable losses in the hatching of pike-perch (Stisostedion 
vitrevm) eggs have frequently made this phase of fish culture a 
ground for investigation. It is, however, not unjust to say that very 
little detailed study has been made and that no definite conclusion 
as to the causes of the high death rate has ever been reached. 

The methods used in the handling of parent fish and eggs are. in 
the main, very much alike at the various stations that hatch pike 
perch. The fishes are caught in nets that are usually pulled once a 
day. weather permitting. Those that are ripe are stripped immedi- 
ately, either in the field or at the hatchery, while the rest are retained 
in pens or live boxes. They are examined in turn daily until found to 
be ripe. In some years the sexes are found to be disproportionate in 
number. If males are scarce, the same individual may be used to 
obtain milt on several .successive days. Fertilization is by the dry 
method, no water, or very little water, being used in the process. Milt 
and eggs are stripped into a bowl in more or less regular alternation, 
and the whole is gently stirred at frequent intervals to insure the con- 
tact of the eggs with the sperm. When the bowl is sufficiently full — 
generally after 10 to 15 minutes — the contents are diluted with water 
which after a varying period is poured off and renewed until the eggs 
are contained in clear water. Cohesion of the eggs, which at this time 
are extremely sticky, is prevented by active stirring or by adding 
silt or starch to the water in addition to such mechanical agitation. 
Finally, after several hours, the eggs are put into the hatching jars 
through which a gentle current of water is kept flowing. 

As already indicated, the losses are very great. Neyin (1887) 
considers a hatch of 50 per cent a very fair success, and this would 
be agreed to by most fish-culturists. The cause of this great mor- 
tality is, in general, ascribed to failure of the eggs to be fertilized 
or else to injuries incurred while the eggs are being handled, espe- 
cially the active stirring and the addition of foreign materials to 
prevent cohesion. It seems almost certain that these last-named 
crude procedures — which so far are unavoidable — are very apt to be 

1 Appendix V to the Report of the U; S. Commissioner of Fisheries for 192L'. B. F. 
Hoc. No. 920. 

91376"— 22 1 



2 U. S. BUREAU OF FISHERIES. 

harmful, and a certain amount of loss is probably due to them. 
Reighard (1890) found that by very careful handling of the eggs at 
and immediately after fertilization the percentage of eggs that- 
started segmentation soon after fertilization could be considerably 
increased. Describing the loss met with in the ordinary course of 
the routine methods, he states that in 252 samples examined 11 per 
cent had died due to lack of impregnation and 33 per cent due to 
injury. Unfortunately, although he designates the day and hour 
at which these observations were made, the age of the eggs is not 
specifically given. From the context it would appear as 29 hours. 
It seems that an egg was designated as dead when it showed an 
opaque white color, a criterion which was adopted also in the present 
investigation. 

The figures of the losses in ordinary handling of pike-perch eggs 
given by Reighard are somewhat at variance with those given by 
L. H. Almy in some unpublished notes on the pike perch. His find- 
ings and those of the authors follow : 



Almy. 



Age of eggs. 



3 hours 

27 to 29 horns 

2 days 

3 days 

4 days 

5 days 

7 days 

9 days 



Per cent 
loss. 



2.5 
5.4 
8.1 
12.6 
31.7 
35.5 
39.0 
33.4 



Sehrader and Schrader 



Age of eggs. 



1 hour 

1 hours 30 minutes 
8 hours 15 minutes 

19 hours 

29 hours 

2 days 

3 days 

4 days 

5 days 



Per cent 

loss. 



0.5 
1.0 
2.4 
4.3 
5.0 
8.3 
13.3 
33.2 
37.1 



It will be seen that in contrast to the 33 per cent of white eggs 
given by Reighard, Almy observed only 5.4 per cent at 27 hours, 
while our own observations are lower still. Almy's and our figures 
agree fairly well, the latter being lower up to two days and a trifle 
higher at four days. It is not quite clear to what such a discrepancy 
could be due, although Reighard's hypothesis of injury as a cause 
of mortality would, of course, itself allow for "large differences on 
account of the varying skill and care bestowed on the eggs. (Tem- 
perature conditions were in all cases apparently the same, the water 
being in the neighborhood of 45° F.) Reighard describes the in- 
jury as taking place most easily over the oil globule, and there is 
no reason to dispute the observation. However, the following ex- 
planation which he advances to account for this phenomenon does 
not seem to rest on a very firm physical basis (Reighard, 1890, pp. 
33, 34) : 

In the natural position the yolk sphere lies with its lower half against the 
egg membranes. These membranes, therefore, support this half of the yolk, 
surrounding it as if it were resting at the bottom of a cup. The upper half 
of the yolk is, on the contrary, not of the same form as the investing membrane; 
its spherical surface is interrupted by the protruding oil globule. 

The result of this arrangement is that when any pressure is brought to bear 
on the egg membranes, so that the space within which the yolk lies is reduced, 
the yolk is able to resist this pressure by fitting itself against the egg mem- 
brane at every part of its surface except over the oil globule. The strain, 



MORTALITY IX PIKE-PERCH EGGS IN HATCHERIES. 6 

therefore, comes on that part of the protoplasmic investment of the yoke which 
covers the oil globule and here it hursts. In almost every case the white spot 
which indicates the rupture of the yoke investment makes its appearance at 
the oil globule, usually at the equator. 

Almy's as well as our own observations show that the death rate 
increases rapidly and steadily to the fourth day and then advances 
more slowly. To begin with, it must be noticed that a small per- 
centage of dead eggs is found practically as soon as the fishes are 



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Fig. 1. — Graph showing the variation in numbers of abnormal and unsegmented pike- 
perch eggs during development. 

stripped. It is surmised that these may have been injured in the 
process of stripping, or that they may have died through some de- 
velopmental irregularity while still in the fish. 

Coming now to the hypothesis that failure of fertilization is re- 
sponsible for a greater part of the mortality of hatching eggs, it is 
generally assumed that lack of impregnation and failure to segment 
are closely correlated. A detailed examination of the material does 
not bear this out. We found that in eggs 4 hours 30 minutes old there 
was a considerable percentage which showed no trace of cleavage. 
This was true also at 5 hours 30 minutes, 6 hours 45 minutes, and 



U. S. BUREAU OF FISHERIES. 



even 8 hours 15 minutes. Most of the unsegmented eggs at the 
latter stages on being; examined cytologically appeared to be normal. 
They were therefore merely lagging- behind. The proportion of 
eggs which has failed to cleave becomes progressively less with age. 
which in itself supports the idea that we are dealing here rather with 
a delay in cleavage than with a lack of impregnation. The curv.e 
in figure 1 (p. 3) shows graphically the numerical conditions en- 
countered. These data are emphasized here merely to show that it is 
manifestly impossible even after eight hours to designate eggs as un- 
fertilized when the absence of segmentation is taken as a criterion. 











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8 9 10 

Figs. 2 to 10. — Surface views of pike-perch eggs. Magnification, approximately X 100. 



Fig. -. — Normal 6-hour 4-celled blastoderm. Fig. 7.- 

FlG. 3. — Normal 19-hour blastoderm. Fig. 8.- 

Fig. 4. — Abnormal 8-hour blastoderm. Fig. 9.- 

Fig. •">. — Abnormal 10-hour blastoderm. Fig. 10.- 
Fi<;. 6. — Abnormal 10-hour bla.stoderm. 



-Abnormal 19-hour blastoderm. 
-Abnormal 19-hour blastoderm. 
-Abnormal 29-hour blastoderm. 
-Abnormal 29-hour blastoderm. 



In addition to all this it must be considered that in the artificial 
insemination of the pike perch the eggs are immersed in milt which 
is diluted very little. The chance of a normal ripe egg remaining 
unfertilized must therefore be extremely small, and, as a matter of 
fact, it is surprising that polyspermy is not more often encountered. 
In addition to the eggs which are found to be dead almost imme- 
diatelv. and to those which are slow to cleave, there is a third class 
Which has been designated as "abnormal." In explanation it must 
be stated that minor irregularities in cleavage are not necessarily an 
indication of pathological conditions (H. V. Wilson, 1891), and only 
such extreme cases as are shown in figures 4 to 10 were rated as 



MORTALITY IX PIKE-PERCH EGGS IN HATCHERIES. 5 

abnormal. Eggs showing- normal cleavage are shown in figures 2 and 
3. Extreme variations, such as are shown in figures 4 to 10. in 
size of cleavage cells were found in nearly all cases to be correlated 
with internal conditions which presaged embryonic death sooner or 





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16 



Figs. 11 to 17. — Sections of pike-perch eggs. Magnification, approximately X 200. 

Fi<;. 11. — Horizontal section of a normal egg of 64 or more cells. 
Fig. 12. — From a 29-hour egg showing cytasters and abnormal spindles. 
Fig. 13. — From a 29-hour egg showing size variation in cytasti 
Fig. 14. — From an 8-hour 15-minute egg, showing elongated nucleus. 
Fig. 15. — From a 29-hour 15-minute egg, showing partial segmentation. 
Fig. 16 — From a 29-hour 15-minute egg showing degeneration in chromatin and multi- 
plication of chromosomes. 

Fig. 17. — From an S-hour 15-minute egg showing monaster. 



later. The number of such abnormal eggs increased steadily with 
age, ranging from 1 per cent at 4 hours 30 minutes to 21 per cent 
at 19 hours. As the curve in figure 1 shows, this increase runs 
parallel with a decrease in the number of unseginented eggs, which 
suggests the possibility that such abnormal cases are derived chiefly 



U. S. BUREAU OF FISHERIES. 



from eggs which are slow to cleave. It may be remarked that it is 
difficult to draw a strict line at times between the abnormal and the 






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Figs. 18 to 22. — Sections of pike-perch eggs. Magnification, approximately X 1620. 

Fig. 18a, 18&, ISc. — Side views of three sections of a plate showing ?.0 chromosomes, 
the normal number. 

Fig. 19. — Side view of a plate in another cell in the same blastoderm as figure 11, 
showing 15 chromosomes, the haploid number. 

Fig. 20. — Polar view of normal plate. 

Fig. 21. — Abnormal multiplication of chromosomes. 

Fig. 22. — Abnormal elongated nucleus — abortive division. 

unsegmented types in the first few hours. At that time one or a few 
minute excrescences are occasionally budded from the germinal disk, 



MORTALITY IX PIKE-PERCH EGGS IX HATCHERIES. 7 

and it becomes a question whether these should be regarded as cells 
or not. 2 On the other hand, at the 29-hour stage, abnormal eggs 
are often segmented into such small cells in such a way that, exter- 
nally, it is very difficult to tell them from normal eggs, although in- 
ternally they may be shown to be very irregular in behavior. The 
intermediate stages are therefore the best material for this phase of 
the investigation. 

Those cytological features in the development of normal eggs 
which bear on the work in hand are as follows : Up to the 16-cell 
stage cell walls are sometimes partially or completely absent, but 
asters and spindles are normal in size and occupy the same position 
that they would if there were a distinct separation into cells. Fol- 
lowing the 16-cell stage, the cleavage, which becomes externally com- 
plete at least in the surface layer of cells, gives evidence of this fact 
internally by the presence of very distinct cell walls. Mitosis is at 
first synchronous in all the cells, but this regularity is soon lost, so 
that certain cells of an egg may be in the resting condition while 
neighboring cells may be undergoing mitotic division. 

As already indicated, cytological examination of uncleaved eggs at 
4 to 8 hours showed the majority to be normal (fig. 11, p. 5). The 
few exceptions were found to have anomalous mitotic figures, and 
their number was increased in the 8-hour stage. At 29 hours every 
uncleaved egg showed anomalous internal features. The exceptional 
8-hour eggs often show a very large monaster (fig. 17, p. 5). Other 
eggs may show several cy tasters and an occasional spindle (figs. 12 
to 16, p. 5). At 29 hours no such large monasters are found in eggs 
of this type or in those called abnormal and generally there is only 
an increase of cytasters in the former. 

The abnormal eggs often present a curious mixture of spindles 
and asters of varying sizes, drawn-out nuclei, chromosomal irregu- 
larities, and partially formed cell walls (figs. 12 to 17, 21, and 22). 
Frequently an egg is found in which a part has undergone regular 
cleavage while the rest is filled with cytasters and shows no indica- 
tion of cell walls (fig. 15). As it was expected that such irregu- 
larities would be reflected in the distribution of the chromosomes in 
division, evidence of such chromosomal abnormalities was sought. 
But, as in other teleosts, the chromosomes are usually so clumped that 
an exact' analysis of them is very difficult. In at least one case, how- 
ever, the metaphase plate in one cell showed close to 30 chromosomes 
(which seems to be the diploid number as obtained from counts in 
normal eggs, fig. 20), while the adjoining cell contained only about 
15 (figs. 18 and 19). This might be explained as a case of partial 
fertilization, in which the sperm has instigated a division of the 
egg nucleus and later has fused with one of the nuclei resulting from 
this first division of the egg nucleus. The fusion nucleus would then 
be diploid and the purely maternal nucleus haploid. _ 

Irregularities in cleavage and mitotic figures practically identical 
with those here described have been obtained experimentally by a 
number of investigators. It will be noted that in every experiment 
of this nature the effect is to induce development with one of the 

s Reighard (1890a) mentions excrescences as occurring in correlation with the flow of 
protoplasm in the formation of the protoplasmic cap. Since the number of eggs showing 
the excrescences mentioned above increases long after the formation j>f the cap, in our 
case, the phenomenon described by Reighard is probably not related to it. 



8 U. S. BUREAU OF FISHERIES. 

parent nuclei absent or in a weakened condition, with both in a 
weakened condition, or with the pronuclei incompatible through 
hybridization. A limited survey of the extensive literature on this 
subject will suffice to show the trend of the work. 

E. B. Wilson (1901) found that in artificial parthenogenesis, where, 
of course, only one of the parent' nuclei is present, there occur such 
abnormalities as the formation of cytasters, the multiplication of 
chromosomes without accompanying cell division, multipolar mitoses, 
and delay in cleavage. The chromosome number in the eggs which 
seem to show normal development is haploid. 

Dungay (1913) weakened or injured sperms of several species of 
invertebrates by means of chemical treatment, heat, or staling, and 
development in eggs fertilized by such sperms resulted in delayed 
cleavage, abnormally sized cells, multipolar figures, and similar 
defects. 

O. Hertwig (1911) and G. Hertwig (1912), among others, have 
described the effect of fertilizing eggs with sperms treated with 
radium. Both authors remarked especially a budding phenomenon 
correlated with delayed development. They also describe drawn-out 
nuclei, multiplication of chromosomes in a nucleus, giant nuclei, and 
cytasters, all almost identical with phenomena which we have de- 
scribed in the abnormal pike-perch eggs. 

C. Packard (1914) found that sperms which had been treated with 
radium may stimulate the eggs to cleave but fail to take part them- 
selves in the subsequent development. When the eggs are "radi- 
ated," they show various irregularities, such as abnormal divisions 
or the failure of pronuclei to unite. 

G. and P. Hertwig (1914) produced similar effects to those already 
mentioned by weakening sperms with methlyn blue among other 
reagents. Still more striking are the phenomena produced by fer- 
tilizing the eggs of teleosts with sperms of another species of teleost. 
The whole list of abnormalities given above was reproduced in such 
development. 

It is not within the province of applied biology to go into a 
theoretical consideration of these phenomena. Suffice it to say that 
the weakening or injury of either sperms or unfertilized eggs will 
produce the same defects in the development of all animals so far 
investigated. Physiological and cytological phenomena identical in 
appearance with those produced experimentally in this way have been 
observed also in abnormally developing pike-perch eggs, and it sug- 
gests itself that the cause of such irregularities is of similar nature. 
In other words, there is a weakening of either sperms or eggs before 
fertilization. 

It is highly improbable that natural conditions should induce a 
state that would cause such a large mortality in the embryos, and it 
becomes almost certain that the injury is incurred during the period 
of the captivity of the fishes. 

As has been said in the introduction, most of the fishes are found 
not to be ready for stripping when first caught. They are therefore 
retained in pens or crates until the reproductive products can be 
obtained from them by stripping; in other words, until they are 
" ripe." The penning of fishes prior to spawning is a practice of 



MORTALITY IN PIKE-PERCH EGGS IN HATCHERIES. 9 

long standing in the handling f pike perch as well as many other 
species. Some of these species are known to stand such confinement 
fairly well, but many show various ill effects, such as hardening of 
the ovaries, wateriness of milt, and low percentage of hatched fry. 
Whatever the cause and physiological process involved, be it abnor- 
mal hydrogen ion concentration due to the crowding of the parent 
fishes, or more directly circulatory and nervous relations, the result 
is a degeneration of eggs and sperms. In the case of the pike perch 
especially the consequent mortality may. of course, fluctuate from 
year to year due to such causes as sudden changes of temperature 
(a sudden change of temperature isi known to materially retard the 
ripening of the fishes in the pens) and weather conditions which 
may prevent pulling the nets and therefore postpone examination of 
the caught fishes. Some specimens do not lay eggs even when ripe 
under such conditions. 

It may be of interest in this connection to give the opinions of men 
who have the supervision of pike-perch hatcheries which are located 
at Constantia, N. Y., Swanton, Vt., Put in Bay, Ohio, and Duluth, 
Minn. Their opinions, given in response to a letter of inquiry, are 







N .- -•.,■•■.• - '... ._■. ;•- ■■■- '•■y / K ■.. •• w •■< «• ,-••■•. 



Fig. 23. — Eggs of Stenotomus prior to maturity, a, Normal egg ; b, egg from a parent 
kept in a tank for two weeks. 

not based on numerical data but are the results of practical obser- 
vation. All of these four superintendents and a fifth, who was 
formerly connected with pike-perch w T ork, agree that the mortality 
of eggs is proportional to the time that the adult pike perch are 
retained in pens and, conversely, that the percentage of hatched eggs 
from fish stripped when taken from the net is much greater than that 
of eggs from penned fish. Four of the men believe that both male 
and female are affected by penning, but that the female is more 
susceptible, while the fifth does not commit himself on this point 
but cautions against using the males more than once, i. e.. on several 
days. 

Bearing more directly on the problem are some experiments made 
by the senior author in connection with some other work. Females 
of the common scuppaug (Stenotomus ehrysops) were netted shortly 
before the spawning period and retained in a tank supplied with a 
continual flow of fresh sea water. Specimen of these impenned fishes 
were dissected at intervals of a few days and the ovaries examined 
histologically. A progressive deterioration of the nearly ripe ova 
was observed, which at the end of two weeks had reached such a stage 
as shown in figure 23. 



10 U. S. BUREAU OF FISHERIES. 

The generalization that the high death rate in pike-perch eggs is 
due to lack of impregnation thus seems to be unwarranted. That the 
present methods of preventing cohesion of the eggs are responsible 
for a certain percentage of the mortality is probable, but they do not 
account for all the loss. On the other hand, it has been shown that 
about 25 to 30 per cent of representative samples of 29-hour eggs show 
abnormalities that must lead to either malformation or death. If an 
average loss is then considered as 50 to 60 per cent (and that is a fair 
estimate), about half of this is due to the agency which manifests 
itself in abnormal development. This cause is in all probability to 
be found in the practice of retaining captured fishes in pens for the 
purpose of permitting eggs and sperms to mature. 



BIBLIOGRAPHY. 

I m'xi.ay. Neil S. 

1913. A study of the effects of injury upon the fertilizing power of sperm. 

Biological Bulletin, Marine Biological Laboratory, Woods Hole. 

Mass., Vol. XXV, No. 4. p. 213-260, Pis. I-II. Press of New Era 

Printing Co., Lancaster, Pa. 
Hertwig, Gunther. 

1912. Das Schicksal des mit Radium bestrahlten Spermachromatins im 

Seeigelei. Eine experimentell-cytologische Untersuchung. Archiv 
fiir mikroskopische Anatomie und Entwicklungsgeschichte, Bd. 79, 
Abt. 2, p. 201-241, 3 Taf. Bonn. 
, und Paula Hertwig. 

1913. Beeiniiussung der mannlichen Keimzellen (lurch chemische Stoffe. 

Archiv fiir mikroskopische Anatomie und Entwicklungsgeschichte, 
Bd. 83, Abt. 2, p. 267-306, 2 Taf. Bonn. 

1914. Kreuzungsversuche an Knochenfischen. Ibid, Bd. 84, Abt. 2, p. 

49-88, 1 Taf. 
Hertwig, Oscar. 

1911. Die Kadiumkrankheit tierischen Keimzellen. Ein Beitrag zur ex- 
perimentellen Zeugungs- und Vererbungslehre. Archiv fiir mikro- 
skopische Anatomie und Entwicklungsgeschichte. Bd. 77, Abt. 2, p. 
1-95+97-164, 4 Tuf.+2 Taf. Bonn. 
Nevin, James. 

1S87. Hatching the wall-eyed pike. Transactions, American Fisheries So- 
ciety, Seventeenth Annual Meeting, p. 14-16. New York. 
Packard, Charles. 

1914. The effect of radium radiations on the fertilization of Nereis. The 
Journal of Experimental Zoology, Vol. 16, No. 1, p. 85-129. 3 pis. 
Philadelphia. 
Reighard. Jacob. 

1890. Experiments in the impregnation of pike-perch eggs. 3 Transactions. 

American Fisheries Society, Nineteenth Annual Meeting, p. 30-36. 
New York. 

1890a. The development of the wall-eyed pike, Stisostedion vitreum Raf. 
A popular introduction to the development of bony fishes. Ap- 
pendix, Ninth Biennial Report, Michigan State Board of Fish Com- 
missioners, Dec. 1, 1888, to Oct. 1, 1890, p. 93-158. Pis. I-X. Lan- 
sing. 

1893. The ripe eggs and the spermatozoa of the wall-eyed pike and their 
historv until segmentation begins. Ibid., Tenth Biennial Report. 
Oct. 1, 1890, to Dec. 1, 1892, p. 89-166, Pis. I-V. 
Wilson. E. B. 

1901. Experimental studies in cytology. I. A cytological study of artificial 
parthenogenesis in sea-urchin eggs. Archiv fiir Entwicklungs- 
mechanik der Organismen, Bd. 12, p. 529-596, 7 Taf., 12 figs. 
Leipzig. 

1901a. Experimental studies in cytology. II. Some phenomena of fertili- 
zation and cell division in etherized eggs. III. The effect on cleav- 
age of artificial obliteration of the first cleavage furrow. Ibid. , 
Bd. 13, p. 353-395, mit Taf. 
Wilson, Henry V. 

1891. The embrvologv of the sea-bass (Serranus atrarius). Bulletin, V. S. 

Fish Commission. Vol. IX. for 1899, p. 209-277. Pis. LXXXVIII- 
CVII, 12 text figs. Washington. 

3 " Presented at the meeting by Hersehel Whitaker and erroneously attributed to him by 
the editor of the Transactions." * (Quotation from Dean's Bibliography of Fishes, Vol. II, 
p. 320, published by the American Museum of Natural History, New York, 1917.) 

11 

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